", "Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA", https://en.wikipedia.org/w/index.php?title=Tetramer_assay&oldid=1011368382, Creative Commons Attribution-ShareAlike License, uses tetrameric proteins to detect and quantify T cells, This page was last edited on 10 March 2021, at 14:39. Activated CD4+ T cells are critically involved in providing cognate help to B cells for the production of protective antibodies and modulate the functions of macroph… (B) Splenocytes of control and 2W1S peptide immunized mice gated on live, CD3+,CD8- CD4+ events, stained with ProT2® H-2 IAb Tetramer. Cells were stained on day 12 and day 22 with DRB1*15:01/NFPYLVAYQATVCARA ProT2® Tetramer. [citation needed], Tetramers that bind to helper T-cells have also been developed. A tetramer assay (also known as a tetramer stain) is a procedure that uses tetrameric proteins to detect and quantify T cells that are specific for a given antigen within a blood sample. Alternatively customers can purchase and conjugate our ProM2® MHC Class II Monomers to any other suitable avidin or streptavidin reagent. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. Most cells in the body express MHC class I molecules, which are responsible for processing intracellular antigens and presenting at the cell's surface. MHC Tetramers Application Notes. 3. The tetramers used in the assay are made up of four major histocompatibility complex (MHC) molecules, which are found on the surface of most cells in the body. Class I MHC molecules are made up of a polymorphic heavy α-chain associated with an invariant light chain beta-2 microglobulin (β2m). Cells stained with β-gal Tetramer were negative. Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice . so it's a good idea to stain with a tetramer carrying a different peptide. Fluorophore tagged streptavidin is added to the bioengineered MHC monomers, and the biotin-streptavidin interaction causes four MHC monomers to bind to the streptavidin and create a tetramer. At 4ºC, very little staining was seen at any tetramer concentration. Not all tetramer-staining T cells increased expression of CD69 in response to peptide challenge (the frequency of CD69 + cells was ∼30–40% of the tetramer binding pool). MHC tetramers can bind to more than one receptor on the target T-cell, resulting in an increased total binding strength and lower dissociation rates. MBL International's new Tetramer Staining Guide is a collection of protocols and data to help make your experiments a success. Τhree donors responding to either HLA-A*0101 restricted pp65 363–373, HLA-A*0201 restricted pp65 495–503, or HLA-B*4401 restricted EBV EBNA3b 657–666, were restimulated with the respective peptides for 3 h in the presence of Brefeldin A. In Current Protocols in Immunology, Altman and Davis caution, “Some MHC tetramers (e.g., H-2 K b) have been observed to stain all CD8 + T cells, presumably via an interaction between CD8 and the α3 domain of the MHC molecule. 112(6): 831-42. COLAK- 2006 520322-Immunology Tetramer Staining • Staining is performed by adding MHC:peptide complexes and anti-CD8 antibodies. MHC Tetramer Staining with Human CD8 Clones Request our new Tetramer Staining Guide Pathway Posters. Based on these data, we recommend 3 hours @ 37ºC with a tetramer They bind to class II MHC molecules, which are only expressed in professional antigen-presenting cells like dendritic cells or macrophages. [13], Another study utilized MHC tetramer complexes to investigate the effectiveness of an influenza vaccine delivery method. This enables us to support our customers fully with their relevant application requirements, helping them to make best use of our technology. J Clin Invest. (2005). A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plot from peptide-stimulated HLA-DRB1*01:01 donor cells. ProImmune has extensive experience in testing ProT2® MHC Class II Tetramers in-house in a variety of applications. If a T-cell's receptor matches the peptide being presented by an MHC molecule, an immune response is triggered. In a separate experiment, it was found that significant staining took place within 30 mins, but longer incubations led to slight increases (figure 2). [2] Novak et al. MHC Tetramer Guided Staining of peptide-specific CD8+ T cells in Peripheral Mononuclear Cells (PBMC) in a 96-well plate format Videos. NIH tetramer core and contain the following peptides: LCMV GP 66–77 DIYKGVYQFKSV and chicken Ova 329 –337 AAHAEINEA. Figure 5. ProT2® staining for CD4+ T cell epitope from Influenza Kramer C. et al. J Immunol. (1999). 166(11): 6665-70. When KT15 was used, OVA-specific tetramer positive cells were observed compared with the negative tetramer, while when 53.6.7 was used, marked non-specific staining was observed in both H-2K b β-galactosidase Tetramer samples clone 53.6.7 is known to have poor compatibility with OVA and other H-2K b Tetramers. This allows for two-color staining with the same tetramer allele, ensuring the highest specificity. Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. In the field of vaccination, the quantification of vector- and antigen-specific responses is of interest, as vector-specific responses … [PubMedID: 11359821]. If a T-cell's receptor … Immuno-Oncology Pathway Scientific Posters. To aid detection of low frequency populations that are beyond the limit of detection of standard direct Class II tetramer staining, expansion [1, 2] or enrichment with magnetic beads [3, 4] may be required; although some very low affinity antigens may not give detectable staining. [2] MHC molecules present peptides to T-cells as a way to communicate the presence of viruses, bacteria, cancerous mutations, or other antigens in a cell. The direct detection of antigen-specific CD4+ T cells has been difficult, due to the low frequency of these T cells (down to 1 in 100,000 white blood cells) and the low functional avidity, caused by low MHC/peptide-TCR affinity, reduced TCR clustering and coreceptor effects. T-cell populations can be tracked over the duration of a virus or after the application of a vaccine. Remove supernatant and... Add 5 µL CD1d tetramer and mix gently. ProT2® Tetramers enable you to study in depth CD4+ T cell immune responses in a wide range of disease areas including autoimmune disease, cancer and infectious diseases. Cell Staining and Flow Cytometric Analysis: Prepare cells of interest ; Prior to performing staining, centrifuge the assembled tetramers in tube or plate at 2500xg for 5 minutes at 4°C. [9], An early application of tetramer technology focused on the cell-mediated immune response to HIV infection. In some occasions a specific interaction between CD8 molecules and tetramers occurs. [3] Thus, MHC tetramers that are bioengineered to present a specific peptide can be used to find T-cells with receptors that match that peptide. (A) Data for HLA-DR7/CMV from Prof Paul Moss group, University of Birmingham, UK, (B) Data for H-2 IAb/2W1S from Dr David Withers group, University of Birmingham, UK. Detecting novel lipid-reactive T cells using CD1d tetramers. D, HLA-specific B cell clones were tested with SAB assays to confirm tetramer specificity used for cell sorting. A broad range of MHC Class II complexes specific for a wide range of infectious diseases such as Influenza, CMV, hepatitis B and C, HIV, EBV, as well as cancer-related epitopes are available as ProT2® Tetramers. 14, 39-44 (2002) MHC tetramer and anti-CD8 antibody staining The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8 + … Custom ProT2® Tetramers for your allele/peptide of interest are available within approximately 6 weeks from ordering. Importantly, … Class II MHC molecules present extracellular antigens, allowing helper T-cells to detect bacteria, fungi, and parasites. Staining differences among human CD8 antibody clones. The usage of blood for analysis, as described here, is minimally invasive and allows continuous monitoring, for example in vaccination studies. Fix cells for 1-4 hours at r.t. in 1% methanol-free formalin in PBS following staining and washing. Count the enriched B cells and determine viability by staining with Trypan blue. Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. Dual tetramers may be of key benefit when blocking does not reduce nonspecific tetramer staining of preimmune B cells. staining of cells not secreting cytokines during this period. Figure 4. © Copyright ProImmune Ltd. 2001-2021. When the tetramers are mixed with a blood sample, they will bind to T-cells expressing the appropriate antigen specific receptor. In situ tetramer staining (IST) can be used to determine the localization, abundance, and phenotype of antigen specific T cells in their native environment. Add 2 x 10 6 cells to 12 x 75mm tubes or a 96-well U-bottom plate. [PubMedID: 16272285]. Proinsulin-specific T regulatory cell may control immune responses in type 1 diabetes: implications for adoptive therapy. Tetramers loaded with Kb-SIINFEKL, Kb-LCMV-GP33, Db-HY-smcy, and I-A b-hCLIP 87 served as negative controls. The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8+ T cells in human blood samples. They can also be used to separate cells for culture, expansion and further study. It has been shown . Titration of KT15 revealed an optimal signal to noise ratio at 0.63 µL/sample for OT-I splenocytes. Tetramer Analysis, Cell Phenotyping, and Intracellular Cytokine Staining. ProT2® Class II MHC Tetramers are delivered quickly with stringent quality control and low batch-to-batch variability. Highly active formulations of a broad range of MHC class I molecules[10] now allows non-experts users to make their own custom peptide-MHC complexes from day-to-day in any lab without special equipment. [1] Novak et al. This may be a result of incomplete peptide distribution and/or specific T cell activation within the 2 h stimulation or may reflect a true inability of some tetramer binding T cells to respond to the dose of antigen used. “Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR” AJT DOI: 10.1111/ajt.15950. ProT2® MHC Class II Tetramer (APC Labeled), ProT2® MHC Class II Tetramer (R-PE Labeled), ProT2® MHC Class II Tetramer (Negative Control), Hartnell F. et al. [1] The tetramers used in the assay are made up of four major histocompatibility complex (MHC) molecules, which are found on the surface of most cells in the body. This enables us to support our customers fully with their relevant application requirements, helping them to make best use of our technology. However, immature, “double-positive” CD4 + CD8 + thymocytes express 10- to 30-fold lower levels of TCR than mature T cells and consequently are refractory to tetramer staining . The availability of new flow cytometers capable of measuring other fluorescent parameters opens the possibility of simultaneous use of tetramers labeled with different … A, Representative example of 3 independent experiments depicting the flow cytometry gating strategy to live single cell sort CD3-CD27+IgD-HLA-DR tetramer double positive B cells from PBMC. The tetramers are labeled with a fluorophore, allowing tetramer-bound T-cells to be analyzed with flow cytometry. For optimal results, please follow our MHC peptide loading protocol and tetramer staining protocols developed to obtain high peptide-MHC complex yields with the easYmer kits and subsequently stain T cells. There are commercially available antibodies, which avoid CD8-tetramer interaction while allowing antigen specific binding. 10.1136/bmjdrc-2019-000873. [14], "The Adaptive Immune Response: T lymphocytes and Their Functional Types", "Visualizing antigen specific CD4+ T cells using MHC class II tetramers", "Invariant natural killer T cells: boon or bane in immunity to intracellular bacterial infections? Decant the supernatant and blot the tube on absorbent pad until the pellet is nearly dry. [9] Helper T-cells or CD4+ T-cells express CD4 co-receptors. Plots on the left show cell samples from a CMV-positive donor; plots on the right show cell samples from a CMV-negative donor. The tetramer staining can be combined with functional assays such as intracellular cytokine staining, proliferation by carboxy fluorescein diacetate succinimidyl ester (CFSE)-based methods, and T-Cell degranulation. C, IgG positive clones were screened with HLA class II Lifecodes Lifescreen Deluxe kit to detect HLA antibody. MBL recommends the use of KT15 for murine CD8 staining. 66). Non-specific staining was eliminated from the plot by gating on CD19 negative cells before plotting CD1d tetramer vs CD3. When anti-CD8 clone KT15 was used, however, CD8 positive cells among OT-I mouse spleen cells were specifically stained only with OVA Tetramer at all antibody concentrations tested. All plots were derived by gating on live, CD19-negative, CD14-negative, CD3-positive lymphocytes. [7][8] Stains using soluble MHC monomers were also unsuccessful due to the low binding affinity of T-cell receptors and MHC-peptide monomers. 2. The modified CRI-p cultured cells were, in turn, subjected to fluorescence-activated cell sorter (FACS) analysis, tetramer staining or T-cell functional assays to quantify the antiviral immunity of HLA-A2 (+) HBV and HCV patients receiving antiviral therapies. HLA-DR1 (DRB1*01:01); DR2 (DRB1*15:01); DR3 (DRB1*03:01); DR4 (DRB1*04:01); DRB1*04:02; DRB1*04:05; DR5 (DRB1*11:01); DR7 (DRB1*07:01); DR9 (DRB1*09:01); H-2 IAb; IAd; IAg7. that clone OKT8 may enhance tetramer staining*. (2020). The present study shows that this approach can also be used to rapidly assess the magnitude of influenza-specific CD8+ T cell epitope dominance across lymph nodes and lungs in a murine model … All plots were derived by gating on live, CD19-negative, CD14-negative, CD3-positive lymphocytes. (2020). All Rights Reserved, From the most experienced commercial providers of MHC multimers, Stringent purification and quality control in line with our ISO:9001 quality system, Proven technology for detecting antigen-specific T cells, Broadest range of catalog items available, Industry leading customer service from immunologists who understand your work. [12] The antigen presenting complex that NKT cells bind to involves CD1 proteins, so tetramers made of CD1 can be used to stain for NKT cells. The CTLs that bound to tetramers were sorted into ELIspot wells for analysis of cytokine secretion. (2020). Fixation of cells is compatible with Tetramer staining but only when fixing samples after Tetramer staining. B6 thymocytes were stained with CD4, CD8 (53-6.7) antibodies, and Kb/OVA tetramer. We have developed dedicated staining protocols for CD4+ and CD8+ T cells to ensure highly specific staining with our Tetramers. The cells were subsequently stained on ice … https://doi.org/10.1002/art.41157, Kramer C. et al. Enriched CD8 + T cells from a CMV sero-positive donor were split into 15 aliquots and stained separately with the 15 CMV tetramer color combinations, washed, mixed together and run as a single sample. (2001). F, Percentage of HLA antibody-producing B cell clones from sorted B cells. Any MHC tetramers that are not bound are washed out of the sample before it is analyzed with flow cytometry. 20. ProT2® MHC Class II Tetramers may be used to study CD4+ T cell immune responses in infectious diseases such as CMV (see figure 1 above) and HCV, and in vaccine studies. [4] Quantification and sorting of T-cells by flow cytometry enables researchers to investigate immune response to viral infection and vaccine administration as well as functionality of antigen-specific T-cells. Centrifuge at 300 x g for 5 min. (2019). [6] Previous attempts to quantify antigen-specific T-cells involved the less accurate limiting dilution assay, which estimates numbers of T-cells at 50-500 times below their actual levels. The study of the CD4+ T-cell activation and effector function is fundamental in the characterization of immune responses to vaccination (1). S4 ). ProImmune has extensive experience in testing ProT2® MHC Class II Tetramers in-house in a variety of applications. Do not attempt to fix samples prior to Tetramer staining. CD4+ memory stem T cells recognizing citrullinated epitopes are expanded in patients with rheumatoid arthritis and sensitive to tumor necrosis factor blockade. Mice were given subcutaneous and intranasal vaccinations for influenza, and tetramer stains coupled with flow cytometry were used to quantify the CTLs specific to the antigen used in the vaccine. [citation needed], This methodology was first published in 1996 by a lab at Stanford University. All plots were derived by gating on live, CD19-negative lymphocytes. 3.3 Tetramer 1. Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens. [9], MHC tetramer molecules developed in a lab can mimic the antigen presenting complex on cells and bind to T-cells that recognize the antigen. [4] Scriba et al. In all cases, tetramer staining was done in PBS with 2 mM EDTA, 2% Fetal calf serum, 0.1% sodium azide, and 1 μg/ml purified anti-CD16 and anti-CD32 (ebioscience) for 1 hr at room temperature. The kit contains 5 groups of HLA class II beads and each data point represents a single bead group. As expected, cytochrome c /I-E k tetramers showed minimal staining of 5C.C7 TCR + CD3 + thymocytes even when used at high concentrations (∼90 nM) ( Fig. This was compared to results of cytotoxic assays and plasma RNA viral load to characterize the function of CTLs in HIV infection. Hence, if a tetramer stain specific for a pathogenic peptide results in a positive signal, this may indicate that the person's immune system has encountered and built a response to that pathogen. Figure 2. 175(10): 6334-43. ProT2® MHC Class II Tetramers can be used together with other Class II Tetramer reagents which customers may already have established in their labs. B, IgG antibody production by the clones was determined by ELISA. Certain anti-murine CD8 clones have been shown to affect tetramer staining 1-6. The left plot shows a population of CD4+ tetramer-positive cells in the upper right quadrant from HCV-positive HLA-DRB1*04:01 donor cells stained with DRB1*04:01/SGIQYLAGLSTLPGNPAIASL ProT2® Tetramer. For each test with 106 total cells, prepare: 50 mL of cold buffer (4–8 °C) 100 µL of cold medium (4–8 °C) 1 mL (or 10 mL; see table below) of warm medium (37 °C). 104(12): R63-7. ProT2® MHC Class II Tetramers are delivered with a choice of standard R-PE or APC labels. Tetramer stains can also be paired with functional assays like ELIspot, which detects the number of cytokine secreting cells in a sample. The right plot shows a population of CD4+ tetramer-positive cells in the upper right quadrant from HCV-positive HLA-DRB1*01:01 donor cells stained with DRB1*01:01/TLLFNILGGWVAA ProT2® Tetramer. Double positive iNKT cells were gated. ProT2® Tetramers are made by ProImmune’s experienced team who have been providing superior MHC reagents for more than 20 years, including our world-leading Pro5® MHC Class I Pentamers. Tetra-mer staining was carried out at room temperature for 1 h using 10 – 20 μg/mL of … Optimal staining was seen with 2 ug/ml tetramer at 37ºC (figure 1). Each dot presents 1 clone and only MFI of bead with tetramer specificity is depicted. [5] Generally, if a person's immune system has encountered a pathogen, the individual will possess T cells with specificity toward some peptide on that pathogen. MHC molecules present peptidesto T-cells as a way to communicate the presence of viruses, bacteria, cancerous mutations, or other antigens in a cell. (2003). Data from Prof Ellie Barnes group, University of Oxford, UK. Characterising HCV specific CD4+ T-cells following viral-vectored vaccination, directly acting anti-virals and spontaneous viral cure. 5‐fold and 45‐fold above standard pMHC tetramer staining without tricks, respectively. ProT2® MHC Class II Tetramers allow you to detect single antigen-specific CD4+T cells accurately by flow cytometry. Work fast, keep the cells cold, use pre-cooled solutions which will [2] Class II MHC tetramer use is becoming more common, but the tetramers are more difficult to create than class I tetramers and the bond between helper T-cells and MHC molecules is even weaker. Escherichia coli are used to synthesize the light chain and a shortened version of the heavy chain that includes the biotin 15 amino acid recognition tag. Add 10 μl of appropriate tetramer suspension … MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen. Figure 1. Allocate less cells when staining NKT cell... Wash cells by adding 1 mL PBS containing 2% fetal calf serum. [PubMedID: 12975468]. Combinatorial pMHC tetramer staining. (A) PBMCs from CMV-positive and CMV-negative donors were stained with DRB1*07:01/PDDYSNTHSTRYVTV ProT2® Tetramer. A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plots from HLA-DRB1*15:01 donor cells. [9], Recent advancements within recombinant MHC molecules have democratised peptide MHC complex formulation and subsequent multimerisation. [3] Day et al. These MHC chains are biotinylated with the enzyme BirA and refolded with the antigenic peptide of interest. Figure 3. J Immunol. Peptide sequences are subject to prior evaluation by ProImmune for binding. ProImmune provides ProT2® Class II MHC Tetramers with responsive, expert technical support and customer service. Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.  https://doi.org/10.1111/ajt.15950, Gliwinski M. et al. [9][11], Natural killer T-cells (NKT cells) can also be visualized with tetramer technology. *Reference: Campanelli R, et al. Resuspend the cell pellet in 1 ml Isolation Buffer. On the x-axis are the specificity of the tetramers used depicted. Tetramer staining was performed in 14 ml round-bottom polystyrene tubes (Corning) in 200 μl T-cell culture medium, following Dasatinib treatment for 10 min at 37 °C (ref. Custom ProT2® Tetramers are available within approximately 6 weeks from ordering. Commercial fixing kits can also be used. E, Percentage of sorted HLA-specific B cells from total memory B cells. This allowed for comparison of the immune response (the number of T-cells that target a virus) in two different vaccine delivery methods. We compared the effects of 10 different anti-CD8 antibody clones with MBL’s MHC tetramer staining. Tetramer staining is a rather straightforward and uncomplicated protocol to analyze phenotype and peptide specificity of a T lymphocyte. ProT2® MHC Class II Tetramer staining. (B–F) Tetramer staining in each of these subsets is indicated (open histogram) with staining of the CD4 SP subset acting as negative control (gray histogram). The results obtained showed that patients with a sustained response had a significantly higher increase in the frequencies of tetramer staining of virus … These data demonstrate the additive nature of these tricks when applied to pMHC … MBL International's new Tetramer Staining Guide is a collection of protocols and data to help make your experiments a success. Request our new Tetramer Staining Guide . Once a peptide sequence is accepted for custom synthesis we will synthesize both the peptide and MHC tetramer reagent. (A) The thymocyte subsets defined by CD4 and CD8 staining are listed as “B” through “F”. HLA-DR-specific memory B cell clones isolated from peripheral blood using ProT2® MHC Class II Tetramers. PBMCs from post HCV-vaccinated donors were stimulated with peptide NFPYLVAYQATVCARA and cultured. All plots were derived by gating on live, CD3-positive lymphocytes. Each dot per … Contrary to ordering class II tetramers from in-house or non-commercial facilities, customers do not need to apply and wait for reagents or supply any peptide. The percentages are indicated in each plot. By using a double coding tetramer staining approach the frequencies of T cells specific for six influenza relevant epitopes (Db-NP 366, Db-PA 224, Db-PB1F2 62, Kb-NS2 114, Kb-PB1 703, I-A b-NP 311) tested were simultaneously identified in lymph nodes and lungs. Catalog ProT2® Tetramers are despatched in 1-2 days for Stock items, and 3 weeks for Available items. A population of CD4+ tetramer-positive cells is visible in the upper right quadrant of the plot from CMV-positive donor cells stained with ProT2® Tetramer. MHC tetramers were developed to present HIV antigens and used to find the percentage of CTLs specific to those HIV antigens in blood samples of infected patients. Immunol. [2][3] Tetramer stains allow for the visualization, quantification, and sorting of these cells by flow cytometry, which is extremely useful in immunology. Centrifuge to pellet (750 × g for 5 min). The optimized manufacturing process for ProT2® Tetramers helps to address these issues by improving reproducibility and sensitivity of CD4+ T cell detection. J Clin Invest. CD4+ T cells play a central role in mediating vaccine immune responses by shaping both the humoral and cellular immunity (2). (2020). Int. If the peptides being presented by MHC class I molecules are foreign—for example, derived from viral proteins instead of the cell's own proteins—the CTL with a receptor that matches the peptide will destroy the cell. Then keep on ice in the dark. Place 2–5 × 105 B cells in each 12 × 75 mm tube, add 1.0 ml of Staining Wash Buffer and mix. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. Fifteen combinations of PE, PE-Cy5, PE-Cy7, and APC streptavidin were used to make separate preparations of CMV tetramer of each color combination. PBMCs from healthy donors were stimulated with peptide PKYVKQNTLKLAT (Influenza A HA 307­­­‑319), cultured for 13 days, and stained with DRB1*01:01/PKYVKQNTLKLAT ProT2® Tetramer. Procedure: Transfer 2-10 million lymphoid cells (PBMC or splenocytes) to a FACS tube. [PubMedID: 10606632]. Plots on the left shows a cell sample that has not been stimulated with peptide; plots on the right show the same cells that have been stimulated with peptide.

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